Synthesis and Bioassay of Antagonists of the Luteinizing Hormone Releasing Hormone Having the Azagly10 Moiety

Karl Folkers*' + , Cyril Y. Bowers+, Wilson B. Lutz*. Klaus Friebel*, Teresa Kubiak*, Bernhard Schircks*. and Georg Rampold* * Institute for Biomedical Research, The University of Texas at Austin, Austin, Texas 78712, + Tulane University School of Medicine, New Orleans, Louisiana 70112 Z. Naturforsch. 37b, 1075-1081 (1982); received March 23, 1982 Antagonist, Luteinizing Hormone Releasing Hormone, Ovulation, Peptides, Azaglycine Seven new analogs of the luteinizing hormone releasing hormone (LHRH), having an Azagly10 moiety, and three corresponding Gly10-analogs were synthesized for bioassay and comparison of inhibitory potencies. This study was toward a possible advantage of the Azagly10 moiety to minimize C-terminal degradation, in vivo. Of the three procedures which were studied to achieve Azagly10-peptides, the reaction of cyanate ion with hydrazides was the most favorable. Variations of substitution in position 1 were also studied. The data from the antiovulatory assay showed that an Azagly10 moiety may not depress activity, and may allow equal or even higher activity than the Glv10 moiety, depending on the analog. [N-Ac-D-Thr1, D-p-Cl-Phe2, D-Trp3-6, Azagly10] LHRH was more inhibitory than the corresponding Gly10-analog. Based on pairs of analogs, the following relationships appeared: (1) N-Ac-D-Thr1 was more effective than N-Ac-jo-Cl-Phe1; (2) The L-configuration of Ala as N-Ac-Ala1- was more effective than the D—; (3) N-Ac-Ala1 appeared more effective than the N-Ac-D-Thr1; (4) D-Trp6 appeared more effective than D-Phe6. In an ultimate clinical use of an antagonist of LHRH to block ovulation, the Azagly10 moiety may be advantageous for limitation of enzymatic degradation.

Introduction important for subsequent studies toward the ulti- The luteinizing hormone releasing hormone mate clinical use of an antagonist of LHRH to (LHRH) functions in the mechanisms of evolution prevent ovulation. and conception. Over a thousand analogs of LHRH have apparently been synthesized, some of which Tab. I. Analogs of LHRH. were significant agonists, super-agonists or antag- onists. 1. [N-Ac-D-^-Cl-Phe1'2,D-Trp3'6,Azagly10]-LHRH We have synthesized and bioassayed a series of 2. [(N-Ac-Pro-Pro)1,D-jo-Cl-Pho2,D-Trp3-6, Azagly10]-LHRH analogs (Table I), most of which have the azaglycine- 3. [(N-Ac-Pro-Pro)1,D-jo-Cl-Phe2,D-Trp3'6]-LHRH amide (Azagly) moiety, I, in position 10. For three 4. [N-Ac-D-Ala1,D-^-Cl-Phe2,D-Trp3-6,Azagly10]- of these analogs, the corresponding peptides having LHRH the glycinamide (Gly) moiety, II, in position 10 5. [N-Ac-Ala1,D-^-Cl-Phe2,D-Trp3'6,Azagly10]- were synthesized for comparison of inhibitory activ- LHRH 6. [N-Ac-D-Thr1,D-p-Cl-Plie2,D-Trp3-6,Azagly10]- ities. LHRH 7. [N-Ac-D-Thr1 ,D-p-Cl-Phe2,D-Trp3.6]-LHRH I, -NH-NH-CO-NHo II, -NH-CH2-CO-NH2 8. [N-Ac-Thr1,D-Phe2,D-Trp3-6,Azagly10]-LHRH The rationale for synthesizing and bioassaying 9. [N-Ac-Thr1,D-Phe2,D-Trp3-6]-LHRH these analogs with Azagly was that the C-terminal 10. [N-Ac-Thr1,D-Phe2-6,D-Trp3,Azagly10]-LHRH might be protected, in vivo, against enzymic degra- dation in contrast to the corresponding Gly10 analogs. Such protection might not be observed by In all of the analogs, the amino acid residues in routine in vivo assays for activity, but might be positions 4, 5, 7, 8, and 9 were identical to those in the same positions of LHRH, i.e., Ser, Tyr, Leu, Arg and Pro. Variations were introduced at posi- * Reprint requests should be sent to Dr. Karl Folkers, tions 1, 2, 3, 6, and 10. N-terminal residues (D-Ala, Institute for Biochemical Research, The University of Texas at Austin, Austin, Texas 78712. L-Ala, D-Thr, D-p-Cl-Phe, L-Thr and the di- 0340-5087/82/0800-1075/$ 01.00/0 peptide L-Pro-L-Pro) were N-acetylated.

Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschung This work has been digitalized and published in 2013 by Verlag Zeitschrift in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der für Naturforschung in cooperation with the Max Planck Society for the Wissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht: Advancement of Science under a Creative Commons Attribution Creative Commons Namensnennung 4.0 Lizenz. 4.0 International License. 1076 K. Folkers et al. • Luteinizing Hormone Releasing Hormone

Variations of the solid phase method of Merrifield method appears to offer advantages for continuation were used to synthesize the peptides. The glycin- of the synthesis of new types of Azagly10-analogs of amide analogs (compounds 3,7, and 9) were prepared LHRH" from benzhydrylamine resins. The azaglvcinamide analogs (compounds 1, 2, 4, 5, 6, 8 and 10) were j I 1-HBT HOAc A N"~CO-NHNH H O ClN-^CO-NHNH-CO-NH. , prepared from chloromethyl Merrifield resins by N C02H CHjCN 2 2 z attaching the penultimate amino acid (Pro) to the 13 resin and completing of the synthesis to the N- terminus. The C-terminal Azagly moiety was then All of the peptides Avere purified by combinations introduced after the peptide was cleaved from the of gel-filtration counter-current distribution (CCD) resin. partition chromatography, and high-performance Two methods for introducing the Azagly residue liquid chromatography (HPLC). were studied. Analogs 4. 6, 8, and 10 were synthe- sized by cleaving the peptide-Merrifield resin with Experimental hydrogen fluoride to give the unprotected N- Amino acid derivatives Avere purchased from acetylated peptide carboxylic acids. Condensation Peninsula Laboratories, San Carlos, California except of the latter with semicarbazide (azaglvcinamide) for D-j9-chlorophenylalanine which Avas proA'ided by by means of N-N'-dicyclohexylcarbodiimide (DCC) the SoutliAvest Foundation for Research and Educa- afforded the peptides for purification. tion. San Antonio, Texas. Alpha amino functions Avere protected by the Boc group except that the Analogs 1,2, and 5 were synthesized by a variation Aoc group AA'as used for arginine. Side chain func- of the azide procedure of Dutta et al. [1] which gave tions Avere protected by Z(Ser,Thr). Tos(Arg) and better yields and fewer difficulties with purification o-Br-Z(Tyr). Benzhydrylamine (BHA) resin AA'as than the method which coupled semicarbazide by purchased from Beckman Bioproducts, Palo Alto. California. DCC, Et3N. CH2C12, and DMF Avere DCC. In this azide procedure, the peptide-Merrifield distilled prior to use. All other chemicals Avere resins were subjected to hydrazinolysis to afford reagent grade. protected peptides as hydrazides. Treatment of these hydrazides with butyl nitrite in acidic dioxan General method of synthesis gave azides which were then allowed to react with The peptides Avere synthesized by the solid phase semicarbazide. The protecting groups were then method using a Beckman Model 990 peptide syn- removed with liquid HF. The conditions of hy- thesizer. Attachment of the first amino acid to a drazinolysis may have possibly resulted in minor loss BHA resin Avas by double DCC-mediated coupling. Attachment of the first amino acid to a chloro- of the N-terminal acetyl group. Phillips [2], and methyl resin AA'as by single coupling of the Boc Schmer and Kreil [3]. have shown that complete amino acid cesium salt. TAVO coupling programs removal of N-acetyl groups by hydrazinolysis is Avere employed for peptide chain elongation. Pro- possible at 100 °C for about 17 h. Ail of the Azagly - gram B Avas identical to Program A except that step 14 was replaced by tAA7o steps, 14 and 15; step 14: containing peptides gave strong positive tests [4] for DMF (3x2 min) and step 15: CH2C12 (3x2 min). hydrazine following complete acid hydrolysis. If a ninhvdrin test [7] indicated an incomplete A new method of introducing the azaglycine acylation of amino groups, a second coupling was performed by a repetition of steps 6 through 14. moiety was extended. Dutta and Morlev [5] reported Sequences resulting from incomplete coupling, even the use of cyanate ion to convert hydrazides to aza- after double coupling Avere terminated by acetyla- amino acid derivatives. In this method, a peptide tion. Acetylation of the N-terminal amino acid hydrazide Avas treated Avith cyanate ion in acidic moiety Avas accomplished by adding 25% acetic solution. We used X-Z-L-proline (11) as a model. anhydride in CHoCL-pyridine in place of steps 10 and 11 of the coupling program. Peptides Avere Condensation of 11 Avith hydrazine by means of removed from BHA resins by treatment with an- DCC in the presence of 1-hydroxybenzotriazole hydrous liquid HF containing ca. 20% anisole for (1-HBT) afforded the N-Z-L-prolvlhydrazide (12), l'h at 0 °C. as described [8], Avhich Avas isolated as the £>-toluene sulfonate salt. TLC was performed on E. Merck silica gel G plates When 12 Avas alloAved to react Avith sodium cyanate Avith detection by fluorescence, chlorine-tolidine spray, and ferric ferricyanide. Values for Rf1, Rf2, in dilute aqueous acetic acid. N-Z-L-prolylaza- Rf3 and A'/4 refer to the systems: 1-butanol-acetic glveinamide (13) Avas formed in high yield. This acid-ethyl acetate-Avater (1:1:3:1), ethanol-Avater 1077 K. Folkers et al. • Luteinizing Hormone Releasing Hormone

(7:3); 2-propanol-lM acetic acid (2:1); and 1- Azide couplings butanol-pyridine-acetic acid-water (50:33:1:40). respectively. Optical rotations were measured on a Synthesis of N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D- Perkin-Elmer Model 141 digital readout Polarimeter. Trp-SerfBzl )-Tyr(BrZ )-D-Trp-Leu-Arg(Tos )- Amino acid analyses were performed on a Beckman Pro-NHNH-CO-NH2: The general procedure of Amino Acid Analyzer Model 119. Samples were Klausner and Bodanszky [13] was followed. To a hydrolyzed with constant boiling hydrochloric acid solution of 0.467 g (0.266 mM) of hydrazide 14 in containing about 1 mg of phenol for 24 h at 110 °C 2.5 ml of DMF at —30 °C, was added 0.22 ml in sealed evacuated tubes. (1.33 mM) of 6 M HCl in dry dioxan, followed by Biological assays for in vitro LH releasing and 0.08 ml of 50% isoamyl nitrite in isoamvl alcohol in vivo antiovulatory activity were carried out. as (0.298 mM, 12% excess). After 30 min at —25 to described [9], with one exception. The in vitro assay of — 30 °C, the solution was cooled to —60 °C. Tri- [N-Ac-Alai.D-jo-Cl-Phe2.D-Trp3-6, Azagly10]-LHRH ethylamine (0.185 ml, 1.33 mM) was added, followed was conducted using a culture of enzymatically by semicarbazide hydrochloride (35.6 mg. 0.319 mM, dispersed anterior pituitary cells according to Vale 20% excess), and additional triethylamine (0.044ml, 0.319 mM). The suspension was stirred at 4 °C for et al. [10]. 24 h then at room temperature for two days. A further 35.6 mg-portion of semicarbazide and DCC couplings 0.044 ml of triethylamine were added, and the mixture was stirred for three days. The product was Synthesis of [N-Ac-D-ThrD-p-Cl-Phe2. precipitated with water and filtered. The same D-Trp2'6. Azagly10[LHRH: To a mixture of 90 mg procedure was used for all three hydrazides. 1 2 3 6 10 of [N-Ac-D-Thr .D-p-Cl-Phe ,D-Trp - .desGly ]- N-Z-L-Prolylhydrazide p-toluenesulfonate 12: To LHRH, 450 mg of semicarbazide, and 5 ml of 598 mg (2 mM) of N-Z-L-proline and 280 mg (2mM) DMF, was added 490 mg af DCC. The mixture was of 1-HBT in 6 ml of CH2C12, was added 458 mg stirred for 28 h at room temperature, treated with (2.4 mM) of DCC. The mixture was swirled for ca. 15 g of ice, and then the mixture was extracted 5 min at room temperature, and filtered into a 4 X with 20 ml portions of ethyl acetate. The organic solution of 1.30 g (39.2 mM) of anhydrous N2H4 in extract was washed with 30 ml of water. Lyophiliza- 8 ml of CH3CN with vigorous magnetic stirring. tion of the combined aqueous extract afforded the After 5 min. the mixture was evaporated to a small crude peptide which was purified by gel filtration in volume on a rotating evaporator. To remove residual 2 M acetic acid on a column of Sephadex G-25 and N2H4, 3 ml of 1-butanol was added and evaporated, by semipreparative HPLC on a column of Bondapak and this step was repeated. Ethyl acetate (5 ml) Ci8 wi;h 50% CH3CN-O.OI M ammonium acetate, was added, and some insoluble material was removed pH 5, as the eluting solvent; 15.2 mg of peptide was by filtration. The filtrate was treated with 380 mg obtained. Analogs 4, 6, 8 and 10 were synthesized of ^-toluenesulfonic acid monohydrate which re- similarly. sulted in a clear solution, from which a crystalline solid rapidly separated. Filtration afforded 773 mg Protected peptide hydrazides (89%) of a solid, m.p. 154-166 °C. Two recrystalliza- tions from methanol/ethyl acetate raised the m. p. to 177.5-180 °C; [a]23 — 31.4 °C (c 1.04 MeOH) TLC To about 1 g of the protected peptide-Merrifield 1 2 3 resin suspended in DMF, was added 0.77 ml of Rf 0.71, Rf 0.63, Rf 0.72, Rf( 0.64. The analytical anhydrous hydrazine, and the mixture stirred 4 d data were in agreement with C20H25N3O6S. at room temperature. The cleaved resin was filtered N-Z-L-Prolylazaglycinamide (13): To 179 mg and washed with DMF. The combined filtrate and (0.41 mM) of 12 in 2 ml of warm water containing washings were evaporated, in vacuo (oil pump), and 0.05 ml of acetic acid, was added 31 mg (0.48 mM) the residue was dried overnight over concd. H2SO4. of sodium cyanate. The solution was kept at room After an ether wash, the residue was dissolved in an temperature for 45 min and at 4 °C for 18 h. Filtra- appropriate solvent. The following hydrazides were tion afforded 101 mg (80%) of 13, 187-189 °C. obtained by using the indicated dissolution and Recrystallization of the product from 2-propanol precipitation solvents [11, 12], raised the m.p. to 187-189.5 °C (reported [1] m.p. 189-190 °C); [a]2)5 -56.6 °C (c 1.44 MeOH). [a]*3 14 N-Ac-D-p -Cl-Phe-D -p - Cl-Phe-D-Trp- —90.6 °C (c 1.42 DMF) (reported [1] [a]2^ —43.6 °C Ser (Bzl )-Tyr( BrZ )-D-Trp-Leu-Arg(Tos )- (c 1.4 DMF)); TLC Rfl 0.75, Rf2 0.73, Rf3 0.73, Pro-NHNH2 (precursor of 1); methanol/ether. RfA 0.69. The analytical data were in agreement with C14H18N4O4. 15 N-Ac-Pro-Pro-D -Cl-Phe-D-Trp- Ser (Bzl )-Ty r (BrZ )-D-Trp-Leu-Arg (Tos )- Pro-NHNHo (precursor of 2); ethanol/ether. Results and Discussion 16 N-Ac-Ala-D -p - Cl-Phe-D-Trp-Ser (Bzl)- T vr (BrZ )-D-Trp-Leu-Arg (Tos )-Pro- Dutta et al. [1] reported the synthesis of some 10 NHNH2 (precursor of 5); DMF/H20. analogs of LHRH which have Azagly moieties. 1078 K. Folkers et al. • Luteinizing Hormone Releasing Hormone

They found that some of their analogs had agonist The general experience of investigators, who have and others had antagonist activities, in certain synthesized analogs of LHRH toward more effective assays. Simpson et al. [14] found [D-Xal(2)6, and potent inhibitors of the release of LH for the Azagly10 ]-LHRH (Nal(2) is 3(2-naphthalene)-ala- control of ovulation, has been that relationships nine) to be one of the most potent known agonists. between sequence and anti-ovulatory activity may Since the Azagly residue in position 10 of antagonists have some generally, but the generality may be might protect the C-terminal from enzymatic limited. The relationships may be applicable to degradation, in vivo, in contrast to enzymic attack restricted groups of sequence changes. On the basis of the naturally occurring Gly10 in LHRH, it was of such generality and limitation, the following considered important to extend the chemical and interpretations can be made of the antiovulatory biological data on Azagly10 analogs of LHRH, and activities of the ten new analogs according to the particularly for analogs which have multiple sub- in vivo data of Table III. stitutions for the amino acids in LHRH. These Analog 1 with the N-Ac-D-p-Cl-Phe1 moiety is substitutions have been based upon the results of compared with analog 6, with the N-Ac-D-Thr1 investigations by several groups of investigators, moiety; the rest of the sequence of both analogs including Ling and Vale [15]; Prasad et al. [16]; is identical, [-D-^-Cl-Phe2, D-Trp3-6, Azagly10]- Coy et al. [17]; Rivier et al. [18]; Channabasavaiah LHRH. The N-Ac-D-Thr1-peptide showed 100% and Stewart [19]. inhibition at a dosage of 25 /ig/rat, but the Our synthesis of seven new Azagly10 analogs X-Ac-^-Cl-Phei-analog was inactive at this dosage, resulted from our use and comparison of three which shows the superiority of N-Ac-D-Thr1 over procedures to introduce the Azagly10 moiety. The N-Ac-^-Cl-Phe1, for this pair. newest procedure involved the use of cyanate ion Analog 2 has the Azagly10 moiety and analog 3 in an acidic medium to convert hydrazides into has the Gly10 moiety, with the rest of the sequence Azagly moieties, and appeared to provide a simpler of both analogs being identical, [(N-Ac-Pro-Pro)1, and better procedure. For a preliminary model, this D-^-Cl-Phe2, D-Trp3-6-]-LHRH. The analog with method was used to synthesize Z-L-prolylaza- Azagly10 showed 60% inhibition at a level of glycinamide (13); the analytical data and melting 50 //g/rat and the Gly10 analog showed 20% inhibi- point of 13 were in agreement, but the optical tion at 25 ^«g/rat. Analog 2 may have contained a rotation was considerably higher than that in data little of the deacetylated peptide. Possibly, the reported by Dutta et al. [1].

Tab. II. Amino acid analytical data0.

Peptides Arg Tyr Leu Pro Ser Ala Thr Gly Phe nh3 pClPhe

1 1.05 0.98 0.99 1.10 1.00 2.3 Oa 1.02 1.02 1.06 3 X 1.01 1.00 0.87 3 0.97 0.96 0.87 3 X 0.99 0.99 0.98 1.27 4b 1.09 0.99 1.01 1.04 0.91 0.96 1.08 5 1.00 1.07 1.03 1.01 0.98 1.06 6 0.95 0.98 1.02 1.11 0.97 0.97 1.95 7 1.03 0.97 0.99 1.13 0.93 0.96 0.99 1.77 8 0.98 1.00 1.02 0.98 0.96 0.89 1.00 1.17 9 0.93 0.95 0.98 1.07 1.04 0.99 1.07 0.95 1.16 10 1.00 0.89 0.98 1.16 1.00 0.94 2 X 0.98 1.16

a This product behaved as a mixture of two peptides, which were difficult to separate. The limitation of sample and lack of feasibility to resynthesize this peptide justified assay of the sample. This sample may have contained a little of the deacetylated peptide; b Purification of this analog was difficult. Eventually, a preparation was obtained which showed two approximately equal peaks by HPLC and two spots by TLC; R[ values of 0.73 and 0.92. The spot having Itf3 0.73 was ninhydrin positive, but the spot of i?f3 0.02 was ninhydrin negative. Since the analysis for amino acids was satisfactory, it was concluded that the peptide with R( 0.73 may be deacetylated and the peptide with Rf 0.02 is the N-Ac-D-Ala1-peptide; c Qualitative tests for hydrazine in the hydrolysate were positive. Tryptophan was generally detected, but not quantitated. Parachlorophenylalanine was detected, but not quantitated. Parachlorophenylalanine and tryptophan were quantitated as a pair using a separate standard containing these two amino acids. 1079 K. Folkers et al. • Luteinizing Hormone Releasing Hormone

Azagly10-analog might be more effective than the same remaining sequence, [-D-/>-Cl-Phe2,D-Trp3-6, Gly10 analog. Azagly10]-LHRH. The N-Ac-D-Thr-analog caused Analog 4, having the N-Ac-D-Ala moiety and 100% inhibition at 25 //g/rat and the M-Ac-Ala1 analog 5 having the N-Ac-Ala1 moiety, have the analog caused 50% inhibition at a level of 6 //g/rat. same remaining sequence, [-D-p-Cl-Phe2,D-Trp3-6, Although there was a 4-fold difference in the test Azagly10]-LHRH. The N-Ac-Ala1 analog elicited levels, the biological data indicate preference of 50% inhibition at a level of 6 //g/rat and the N-Ac-Ala over N-Ac-D-Thr for position 1 in this N-Ac-D-Ala1 analog exhibited 14% inhibition at pair of analogs. a level of 6.5 //g/rat. This comparison indicates that Analog 6 has the Azagly10 moiety and analog 7 the L-configuration of Ala in position 1 is superior has the Gly10 moiety, but both analogs are other- to the D-configuration. Analog 4 may have con- wise identical, having [N-Ac-D-Thr1, D-p-Cl-Phe2, tained a little of the deacetylated peptide. D-Trp3-H-LHRH. The Azagly10 analog caused Analog 5, with the N-Ac-Ala1 moiety, and analog 50% inihibition at a level of 6.5 /

Table III. Antiovulatory activities.

Analog Dose Rats No. of ova/SEM Inhibition //g/rat ovulating/treated ovulating rat (i) %

1 25 6/6 12.3 ± 0.7 0 Control - 5/5 12.4 ± 1.1 0 o 50 2/5 4.4 ± 2.8 60 Control - 5/5 12.4 ± 1.1 0 8 25 4/5 9.6 2.5 20 4 25 2/6 3.8 ± 2.5 67 6.5 6/7 11.9 ± 2.0 14 Control - 11/11 13.4 ± 0.6 0 5 6 4/8 5.6 ± 2.4 50 - 6/6 11.5 -j- 1.5 6 25 0/6 0 100 12 3/7 5.4 ± 2.6 57 6.5 5/10 6.8 ± 2.3 50 Control - 11/11 13.4 ± 0.6 0 7 25 1/5 0.8 ± 0.8 80 6.25 4/5 8.4 ± 2.3 20 Control - 8/8 11.9 7 0 8 200 0/11 0 100 Control - 15/15 12.5 4- 0.72 0 50a 3/6 4.3 ± 2.3 50 50b 1/6 0.17 4- 0.17 83.3 50° 1/8 1.3 + 1.2 87.5 Control - 5/5 9.0 — 1.3 0 9 200 1/7 1.28 ± 1.28 86 100 2/9 1.8 ± 1.3 77.8 50 4/9 5.2 rb 2.1 55.6 Control - 6/6 13.16 ± 1.11 0 200'1 0/6 0 100 Control - 5/5 10.6 ± 0.7 0 10 100 0/6 0 100 50 7/10 7.1 ± 1.9 30 Control - 5/5 9.0 ± 1.3 0

a Dispersed in medium 30-40 min before injection; b dispersed in medium 00-90 min before injection; c dis- persed in medium 195 min before injection; (1 reassaved after 3 months.

1081 K. Folkers et al. • Luteinizing Hormone Releasing Hormone procedure of Vale et al. [10], which uses cultures of It is evident that the Azagly10 moiety in compari- dispersed pituitary cells. Analogs 1,3, and 7 at levels son with the Gly10 moiety in analogs which are of 1 /ug significantly (p <0.05-0.001) inhibited the otherwise identical does not cause a significant release of LH. Analogs 3, -1, and 6 significantly reduction in activity for inhibition of ovulation. (p <0.001) inhibited at levels of 3 ,ug. Analogs 8, 9, The Azagly10 analogs are apparently equal to the and 10 showed significant (p <0.01-0.001) inhibition Gly10 analogs in activity, and perhaps may be at levels of 10, 30 and 10 ug, respectively. desirable to study the metabolic stability of Azagly10 As a group, these nine analogs were more in- analogs under conditions which more closely simu- hibitory for the release of LH than for FSH, since late potential clinical use. analogs 1, 2, 3, and 7 did not inhibit release of FSH at levels effective for inhibition of the release of LH, Appreciation is expressed to Dr. Marvin Karten, but analogs 4, 6. 8, 9, and 10 did inhibit release of and to NIH, NICHD Contract NO l-HD-O-2833, FSH at the same level which inhibited LH. By the and to the Robert A. Welch Foundation for their culture of dispersed pituitary cells, analog 5, at a respective support. We are grateful to Drs. Albert Parlow for the NIAMDD program, NIH; Gordon level of 0.02 nanograms, significantly (p <0.001) Niswender; and L. E. Reichert for their RIA inhibited the release of LH. preparations and procedures.

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